The assay will take approximately one hour to stain the cells. In addition, the cells must be paraformaldehyde/ethanol fixed at least one day before staining. The purpose of this is to cross link the DNA so it will not wash out during the staining process. When cells stained with the Apo-BrdU kit are run on the flow cytometer, a dual parameter data display needs be created. One parameter will be apoptosis and the second parameter will be DNA content. This is one of the advantages of the TUNEL assay because not only does it tell you if your cells are apoptotic, but it also tells you where in the cell cycle the cells are apoptotic. No other apoptosis assay does this.

A landmark of cellular self-destruction by apoptosis is the activation of nucleases that degrade the higher order chromatin structure of the DNA into fragments of 50 to 300 kilobases and subsequently into smaller DNA pieces of about 200 base pairs in length. The most consistent hallmark of the apoptotic process, the break-up of the genomic DNA by cellular nucleases, is also one of the easiest and most straightforward to measure (PFS kits take advantage of this fact). As the DNA strands are broken by the nucleases, a large number of 3'-hydroxyl ends of these strand fragments become exposed. This property can be used to identify apoptotic cells by labeling the 3'-hydroxyl ends with bromolated deoxyuridine triphosphate nucleotides (BrdUTP) in the case of the APO-BrdU kit or FITC-dUTPs in the case of the APO-Direct kit. The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes a template independent addition of the dUTP's to the 3'-hydroxyl ends of double- or single-stranded DNA to blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the apoptosis reagent kits from PFS. It has also been established that BrdUTP is more readily incorporated into the DNA break sites of apoptotic cells than are the deoxynucleotide triphosphates complexed to larger ligands like fluorescein, biotin or digoxigenin. This greater incorporation gives rise to the binding of a larger number of fluoresceinated anti-BrdU antibodies to these sites and thus a substantially stronger fluorescent signal from cells labeled in this manner. Below is an illustration of the TUNEL Assay used in the APO-BrdU kit.